1-Substituted-4-pyrrolidinopiperidines as inhibitors of interleukin 1

ABSTRACT

There are disclosed compounds of the formula: ##STR1## wherein R is phenyl, naphthyl, pyridyl, quinolinyl, pyrazinyl, pyrimidinyl, quinoxalinyl quinazolinyl or any of the foregoing substituted with halo, lower alkyl, lower alkenyl, lower alkoxy, carboxy, lower alkoxycarbonyl, hydroxy, amino, mono- or diloweralkylamino, mercapto, lower alkylthio, lower alkylsulfonyl, cyano, nitro or trifluoromethyl; which, by virtue of their ability to inhibit interleukin 1, are of use as antiinflammatory agents and in treatment of disease states involving enzymatic tissue destruction.

This invention relates to novel compounds possessing interleukin 1(IL 1) antagonist activity and having antiinflammatory activity.

Interleukin 1 (IL 1) is a peptide hormone exhibiting a number of immuneand inflammatory actions [Dinarello, Rev. Inf. Dis. 6, 51 (1984)]. II 1is produced, in response to inflammatory stimuli, by leukocytes such asmacrophages and polymorphonuclear cells, as well as by a variety ofother cell types such as synovial cells, endothelial cells andkeratinocytes, and it mediates several biological responses ofleukocytes on other tissue targets such as bone, articular joints,liver, hypothalamus, and brain.

IL 1 was originally shown to augment the proliferation of T lymphocytesfor which it was named lymphocyte activating factor (LAF), and isbelieved to be important for the generation of T cell-dependent immuneresponses.

There is evidence to suggest a relationship between IL 1 and pathologyin various diseases, particularly immunoinflammatory disorders such asrheumatoid arthritis [Dinarello et al., Ann. Rev. Med. 37, 173 (1986)].IL 1 induces acute inflammatory responses producing soft tissue swelling(edema and erythema) [Granstein et al., J. Clin. Invest., 77, 1010(1986)]. It is a chemoattractant for polymorphonuclear leukocytes (PMN)and induces the activation and migration of these cells into tissues. IL1 also stimulates the production of prostaglandin E₂, a potentinflammatory arachidonic acid metabolite, by a variety of cells andtissues including chondrocytes and synovial cells [Mizel et al., Proc.Nat'l. Acad. Sci., 78, 2474 (1981) and Chang et al., J. Immunol., 136,1283 (1986)] and hypothalamic tissue. This effect on the hypothalamus isthought to be responsible for fever production. IL 1 can inducearticular joint destruction by stimulating the production of a varietyof hydrolytic enzymes (neutral proteases such as collagenase,glycosaminoglycanases, etc.) which degrade cartilage matrix proteins(collagen, proteoglycan, etc.) by synovial cells, chondrocytes, andfibroblasts [Dayer et al., Science, 195, 181 (1977) and Postlethwaite etal., J. Exp. Med., 157, 801 (1983)]. Furthermore, IL 1 induceshyperproliferation of dermal and synovial fibroblasts and is a potentinducer of bone resorption [Wood et al., J. Immunol., 134, 895 (1985)and Gilman and Kimball, Agents and Actions, 16, 468 (1985)].

Finally, IL 1 mediates acute phase reactions including alterations inplasma divalent cations, increased synthesis by liver cells of acutephase proteins (C-reactive protein, serum amyloid A, etc.) and fever.Accordingly, compounds which have IL 1 antagonist activity and therebyinhibit the biological effects of IL 1 can be advantageously used toblock pathologies in which one or more of these events occur such asrheumatoid arthritis, osteoathritis and related disorders [Rodman andSchumacher, eds, "Primer on the Arthritic Diseases" 8 ed. Atlanta,1983], psoriasis and other inflammatory/proliferative skin disorders aswell as diseases in which the secretion of collagenase (and other tissuehydrolysing neutral proteinases) has been implicated as a causativefactor, including periodontal disease, tumor invasiveness, andepidermolysis bullosa [Perez-Tamayo, Amer. J. Pathol., 92, 509 (1978)and Harris and Krane, N. Engl. J. Med., 291, 652 (1974)] and so forth.

It has now been found that certain novel1-substituted-4-pyrrolidinopiperidines antagonize the activity of IL 1,and so are useful as antiinflammatory agents and in the treatment ofpathologies whose etiology is collagenase-based tissue destruction. Thepresent invention provides novel compounds having the formula: ##STR2##wherein R is phenyl, naphthyl, pyridyl, quinolinyl, pyrazinyl,pyrimidinyl, quinoxalinyl, quinazolinyl or any of the foregoingsubstituted with halo, lower alkyl, lower alkenyl, lower alkoxy,carboxy, lower alkoxycarbonyl, hydroxy, amino, mono- ordiloweralkylamino, mercapto, lower alkylthio, lower alkylsulfonyl,cyano, nitro or trifluoromethyl.

The terms "lower alkyl", "lower alkenyl" and "lower alkoxy" refer inmoieties having 1 to 6 carbon atoms in the carbon chain.

The especially preferred compounds are those having the formula:##STR3## wherein R is 4-cyanophenyl or 4-(methylsulfonyl)phenyl.

The compounds of the invention can be prepared by several routes.According to one route, 1,4-dioxa-8-azaspiro[4.5]decane is reacted witha suitable halo-R reactant, and following ketal hydrolysis, theresultant intermediate is reacted with pyrrolidine in the presence of anacid, such as p-toluenesulfonic acid to yield an intermediatepyrrolidino tetrahydro pyridine: ##STR4## In the final step, theintermediate is reduced with a suitable reducing agent, such as, forexample, tetrabutylammonium cyanoborohydride to yield the desired finalproduct: ##STR5##

The starting materials used in the above outlined preparative sequencesare all available commercially or can be prepared by conventionalmethods disclosed in the chemical literature.

The compounds of the invention, by virtue of the ability to antagonizeinterleukin 1, are useful in the treatment of such diseases asrheumatoid arthritis, osteoarthritis, tendinitis, bursitis and similarconditions involving inflammation, as well as psoriasis and otherinflammatory/proliferative skin disorders. Moreover, the compounds areuseful in treating disease states involving enzymatic tissuedestruction, for example, conditions in which collagenase has beenimplicated as a causative factor, such as rheumatoid arthritis jointdestruction, periodontal disease, tumor invasiveness, cornealulcerations, epidermolysis bullosa and the like.

When the compounds of the invention are employed as antiinflammatoryagents, or collagenase inhibitors, they can be formulated into oraldosage forms such as tablets, capsules and the like. The compounds canbe administered alone or by combining them with conventional carriers,such as magnesium carbonate, magnesium stearate, talc, sugar, lactose,pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, low melting wax, cocoa butter and the like.Diluents, flavoring agents, solubilizers, lubricants, suspending agents,binders, tablet-disintegrating agents and the like may be employed. Thecompounds may be encapsulated with or without other carriers. In allcases, the proportion of active ingredients in said compositions bothsolid and liquid will be at least to impart the desired activity theretoon oral administration. The compounds may also be injected parenterally,in which case they are used in the form of a sterile solution containingother solutes, for example, enough saline or glucose to make thesolution isotonic. For topical administration, the compounds may beformulated in the form of dusting powders, solutions, creams, lotions oraerosols in pharmaceutically acceptable vehicles, which are applied toaffected portions of the skin.

The dosage requirements vary with the particular compositions employed,the route of administration, the severity of the symptoms presented andthe particular subject being treated. Treatment will generally beinitiated with small dosages less than the optimum dose of the compound.Thereafter the dosage is increased until the optimum effect under thecircumstances is reached. In general, the compounds of the invention aremost desirably administered at a concentration that will generallyafford effective results without causing any hamful or deleterious sideeffects, and can be administered either as a single unit dose, or ifdesired, the dosage may be divided into convenient administered atsuitable times throughout the day.

The interleukin 1 antagonist activity, as well as the antiinflammatoryeffects of the compounds of the invention may be demonstrated bystandard pharmacological procedures, which are described more fully inthe examples given hereinafter.

These procedures illustrate the ability of the compounds of theinvention to inhibit the IL 1-induced release of neutral protease fromarticular chondrocytes.

The following examples show the preparation and pharmacological testingof compounds within the invention.

EXAMPLE 1 4-[4-(1-Pyrrolidinyl)-1-piperidinyl]benzonitrile (A)4-(1,4-Dioxa-8-azaspiro[4.5]dec-8-ylbenzonitrile

A mixture of 10 g (0.0825 mol) p-fluorobenzonitrile, 47 g (0.3282 mol)of 1,4-dioxa-8-azaspiro[4.5]decane, 17 g (0.123 mol) of K₂ CO₃, and 100ml of acetonitrile is stirred at 90°-100° C. for three days. Thereaction mixture is allowed to cool to ambient temperature, diluted withwater and extracted with methylene chloride. The combined extracts arewashed with brine, dried over Na₂ SO₄, and concentrated in vacuo to givea pasty solid. Trituration with ethyl ether furnishes 13.4 g (67%) oftitle compound: m.p. 134°-135° C.; IR (KBr) 2210 and 1600 cm⁻¹ ; NMR(CDCl₃) δ7.48 (d, 2H), 6.88 (d, 2H), 4.0 (s, 4H), 3.58-3.40 (m, 4H), and1.90-1.70 (m, 4H).

(B) 4-(4-Oxo-1-piperidinyl)benzonitrile

A mixture of 12 g (0.049 mol) of the ketal of (A), 120 ml of 10% H₂ SO₄solution, and 60 ml of tetrahydrofuran is stirred at ambient temperaturefor 4 days. The reaction mixture is diluted with water and extractedwith methylene chloride. The combined organic extracts are dried overNa₂ SO₄ and concentrated in vacuo to give a pasty solid. Triturationwith ethyl ether provides 4.5 g (46%) of the compound: m.p. 100°-101°C.; IR (KBr) 2220 and 1700 cm⁻¹ ; NMR (CDCl₃) δ7.54 (d, 2H), 6.90 (d,2H), 3.88-3.66 (m, 4H), and 2.70-2.52 (m, 4H).

(C) 4-(3,6-Dihydro-4-(1-pyrrolidinyl)-1(2H)-pyridinyl]benzonitrile

A solution of 24 g (0.1199 mol) of the compound of (B), 15 ml (12.78g/0.1796 mol) of pyrrolidine, 240 mg of p-toluenesulfonic acid, and 100ml of benzene is stirred at reflux with the azeotropic removal of waterfor 6 hours. After cooling, the resulting precipitate is collected andtriturated with ethyl ether to afford 20 (66%) of title compound: m.p.126°-129° C.; IR (KBr) 2200, 1635, and 1600 cm⁻¹ ; NMR (CDCl₃) δ7.48 (d,2H), 6.7 (d, 2H), 4.3-4.1 (m, 1H) 3.95-3.45 (m, 4H), 3.2-2.85 (m, 4H)2.65-2.25 (m, 2H), and 1.95-1.75 (m, 4H).

Analysis for: C₁₆ H₁₉ N₃ : Calculated: C, 75.85; H, 7.56; N, 16.59.Found: C, 75.42; H, 7.51; N, 16.57.

(D) 4-[4-(1-Pyrrolidinyl)-1-piperidinyl]benzonitrile

A solution of 2 g (0.0079 mol) of the compound of (C), 5 ml of 4.66M HCl(in isopropyl alcohol), 40 ml of methylene chloride and 4.46 g oftetrabutylammonium cyanoborohydride is stirred at ambient temperatureovernight. The solution is diluted with sat. Na₂ CO₃ solution andextracted with methylene chloride. The combined extracts are dried (Na₂SO₄) and concentrated in vacuo to give the title compound. An analyticalsample is prepared by recrystallization from cyclohexane: m.p. 88°-89°C.; IR (KBr) 2200, 1600, and 820 cm⁻¹ ; NMR (CDCl₃) δ7.45 (d, 2H), 6.85(d, 2H), 4.0-3.7 (m, 1H), 2.95-2.65 (m, 8H), and 2.15-1.65 (m, 8H); M.S.m/e 256 (M+1).

Analysis for: C₁₆ H₂₁ N₃.1/2H₂ O: Calculated: C, 72.69; H, 8.39; N,15.89. Found: C, 72.34; H, 7.91; N, 15.72.

EXAMPLE 2 1-[4-(Methylsulfonyl)phenyl]-4-(1-pyrrolidinyl)piperidine (A)8-[4-(Methylsulfonyl)phenyl]-1,4-dioxa-8-azaspiro[4.5]decane

A mixture of 10 g (0.057 mol) of p-fluorophenyl methyl sulfone, 8.72 g(0.063 mol) of K₂ CO₃, 24.66 g (0.172 mol) of1,4-dioxa-8-azaspiro[4.5]-decane and 50 ml of acetonitrile is stirredovernight at 90°-100° C. After cooling, the mixture is diluted withwater and extracted with methylene chloride. The combined extracts arewashed with brine, dried over Na₂ SO₄, and concentrated in vacuo.Trituration with ethyl ether affords 13.11 g (77%) of title compound asa white solid: m.p. 192°-194° C.; IR (KBr) 1588, 1369, and 1290 cm⁻¹ ;NMR (CDCl₃) δ7.74 (m, 2H), 6.96 (m, 2H), 4.02 (s, 4H), 3.54 (m, 4H),3.03 (s, 3H), and 1.81 (m, 4H).

Analysis for: C₁₄ H₁₉ NO₄ S: Calculated: C, 56.54; H, 6.44; N, 4.71.Found: C, 56.37; H, 6.55; N, 4.97.

(B) 1-[4-(Methylsulfonyl)phenyl]-4-piperidinone

A mixture of 12.97 g (0.0436 mol) of the ketal of (B) and 200 ml of 10%H₂ SO₄ /tetrahydrofuran (2:1) solution is stirred at 60°-70° C. for 4hrs and is then allowed to stand at room temperature for 3 days. Themixture is diluted with water and extracted with methylene chloride. Thecombined extracts are washed with brine, dried over Na₂ SO₄, andconcentrated in vacuo. Trituration with ethyl ether gives 10.07 g (91%)of title compound as a white solid: m.p. 183°-185° C.; IR (KBr) 3410,1710, 1585, and 1160 cm⁻¹ ; NMR (CDCl₃) δ7.79 (d, 2H), 6.98 (d, 2H),3.79 (t, 4H), 3.04 (s, 3H), and 2.61 (t, 4H).

Analysis for: C₁₂ H₁₅ NO₃ S₄ : Calculated: C, 56.89; H, 5.97; N, 5.53Found: C, 57.36; H, 6.23; N, 5.88.

(C)1,2,3,6-Tetrahydro-1-[4-(methylsulfonyl)phenyl]-4-(1-pyrrolidinyl)pyridine

A solution of 24 g (0.0947 mol) of the compound of B), 12 ml (10.2g/0.143 mol) of pyrrolidine, 240 mg of p-toluenesulfonic acid and 150 mlof benzene is heated at reflux with the azeotropic removal of water and6 hours. After cooling the resulting precipitate is collected andtriturated with ethyl ether to afford 22.5 g (78%) of title compound:m.p. 141°-143° C.; IR (KBr) 1650, 1580; 1300, and 1130 cm⁻¹ ; NMR(CDCl₃)δ7.75 (d, 2H), 6.85 (d, 2H), 4.30-4.10 (m, 1H), 3.95-3.45 (m,4H), 3.25-2.85 (m, 4H), 3.05 (s, 3H), 2.65-2.30 (m, 2H), and 1.95-1.70(m, 4H).

Analysis for: C₁₆ H₂₂ N₂ O₂ S: Calculated: C, 62.71; H, 7.24; N, 9.14.Found: C, 62.46; H, 7.04; N, 9.01.

(D) 1-[4-(Methylsulfonyl)phenyl]-4-(1-pyrrolidinyl)piperidine

A solution of 2 g (0.0065 mol) of the compound of (C), 5 ml of 4.66M HCl(in isopropyl alcohol), 40 ml of methylene chloride and 2.68 g oftetrabutylammonium cyanoborohydride is stirred at ambient temperatureovernight. The solution is diluted with sat. Na₂ CO₃ solution andextracted with methylene chloride. The combined extracts are dried (Na₂SO₄) and concentrated in vacuo to give 1.6 g (80%) of the titlecompound. An analytical sample is prepared by recrystallization fromethyl acetate: m.p. 166°-170° C.; IR (KBr) 1595, 1295, and 880 cm⁻¹ ;NMR (CDCl₃)δ7.75 (d, 2H); 6.9 (d, 2H), 4.0-3.7 (m, 1H), 3.15-2.55 (m,8H), 3.0 (s, 3H), and 2.05-1.55 (m, 8H)

Analysis for: C₁₆ H₂₄ N₂ O₂ S: Calculated: C, 62.30; H, 7.84; N, 9.08.Found: C, 62.40; H, 7.41; N, 8.85.

EXAMPLE 3

The ability of the compounds of the inventions to inhibit interleukin 1is measured by the ability of the test compounds to inhibit the IL1-induced release of neutral protease from rabbit articularchondrocytes.

This assay is carred out as follows:

Isolation of rabbit chondrocytes

Male New Zealand White rabbits are anesthetized with 50 mg/kg ofketamine (i.m.) and killed by an intracardiac injection of 3 mls. ofNembutal. The knee joints of both legs are resected and the articularsurfaces are exposed. Cartilage slices are obtained using scalpel andare placed in a tissue culture dish (100 mm diameter) containing 10 mlsof Hank's balanced salt solution (HBSS). The chondrocytes within thecartilage slices are then liberated by a series of enzyme digestions.The slices are incubated for 10 min. at 37° C. in 0.05% hyaluronidase(Sigma H-3884), rinsed with HBSS and incubated with 0.2% trypsin (SigmaT-2395) for 10 min. at 37° C. The slices are rinsed again and incubatedfor 10 mins. at 37° C. with 1.2% collagenase (Sigma C-5138). The slicesare then rinsed again with HBSS and resuspended in 10 ml of Ham's F-12medium containing 10% fetal bovine calf serum (FCS) and 0.2% collagenaseand incubated overnight at 37° C. in a 5% CO₂ incubator. The next day,the medium containing the digested cartilage fragments and liberatedchondrocytes is transferred to a 15 ml centrifuge tube and the cells arecollected by centrifugation and washed twice and resuspended in Ham'sF-12 medium. The cells are then plated into 24-well tissue cultureplates (2×10⁵ cells/well) and incubated at 37° C. until confluent(usually 4-6 days).

Stimulation of chondrocytes and drug treatment

The confluent chondrocytes are rinsed twice with serum-free Ham's F-12medium and 1 ml is added to each well. Fifty μl of purified human IL 1(100 Units/ml; Genzyme Corporation, Boston, MA) is then added tostimulate these cells to secrete neutral protease. To measure drugeffects, the cells are treated with test compound 10 min. prior toaddition of IL 1. The standard screening dose is 10 μM. Twenty-fourhours after IL 1 stimulation, supernatant fluids are collected andassayed for neutral protease activity.

Neutral protease assay

The neutral protease activity of chondrocyte supernatant fluids isdetermined by their ability to degrade an insoluble protease substrate,azocoll (Sigma). Supernatants are treated for 10 min. at roomtemperature with 350 μM p-aminophenylmercuric acetate to activate thelatent enzyme. Three hundred μl of supernatant is then mixed with 500 μlof a 20 mg/ml suspension of azocoll and incubated at 37° C. for 18-24hrs. with gentle rocking. The mixtures are centrifuged and the amount ofsubstrate hydrolyzed is determined by measuring the absorbance of thesupernatant at 520 nm.

Drug effects are calculated as the % change in enzyme activity(absorbance) by supernatants from drug-treated chondrocytes relative toenzyme activity of supernatants from vehicle-treated chondrocytes asfollows: ##EQU1##

Where tested in this assay, the compounds of the invention gave thefollowing results:

    ______________________________________                                        Compound of     Dose    % Inhibition                                          Example No.     (μM) (I.S.D.)                                              ______________________________________                                        1               10      26                                                                    1       57                                                                    0.1     48                                                    2               10      91                                                                    1       87                                                                    0.1     22                                                    ______________________________________                                    

The results show that the compounds tested exhibit a moderate to verysignificant inhibition of IL 1-induced protease secretion.

What is claimed is:
 1. A compound having the formula: ##STR6## wherein Ris phenyl or naphthyl or any of the foregoing substituted with halo,lower alkyl, lower alkenyl, lower alkoxy, carboxy, lower alkoxycarbonyl,hydroxy, amino, mono- or diloweralkylamino, mercapto, lower alkylthio,lower alkylsulfonyl, cyano, nitro or trifluoromethyl.
 2. The compound ofclaim 1, having the name4-[4-(1-pyrroldinyl)-1-piperidinyl]benzonitrile.
 3. The compound ofclaim 1, having the name1-[4-(methylsulfonyl)phenyl]-4-(1-pyrrolidinyl)piperidine.